Carcinogenesis is a multistep process initiated by DNA damage, gene mutation, gene rearrangement and gene translocation, and ending with phenotypic transformation of cancer cells. Bishop, Science 235:305-311 (1987).
It is well documented that normal cellular genes ("proto-oncogenes") can be converted into oncogenes or cancer genes due to agents which cause DNA strand breaks and damage. Kasid, et al. Carcinogenesis, 7:327-330 (1986). Often, conversion involves the breakage of the proto-oncogene from DNA and its movement and placement through another break into another chromosome. Also, it is known that proto-oncogenes can become activated to become malignant oncogenes by gene amplification. Stark et al., Ann. Rev. Biochem. 53:447-491 (1984).
Most living cells possess systems tier recognizing and eliminating DNA damage. For example, the prokaryote, E. coli, possesses a variety of enzymes for responding to DNA damage. Such enzymes include those of the SOS repair system and various Rec proteins. These enzymes, and others, respond to DNA damage caused by U.V. radiation, chemical mutagens and the like. Eukaryotic and mammalian cells also possess DNA repair enzymes.
One mammalian enzyme, poly (ADP-ribose) polymerase, appears to play an important role in recovery of cells from DNA strand-breaking events. It has been reported that poly (ADP-ribose)polymerase activity is higher in isolated nuclei of SV 40 transformed fibroblasts than in those of untransformed fibroblasts; that leukemic cells showed higher enzyme activity than normal leukocytes; and that colon cancers showed higher enzyme activity than normal colon mucosa. Miwa et al., Arch. Biochem. Biophys. 181:313-321 (1977); Burzio et al., Proc. Soc. Exp. Biol. Med. 149:933-938 (1975); Hirai et al., Cancer Res. 43:3441-3446 (1983). It was concluded in these reports that the activity of the poly (ADP-ribose) polymerase responds to DNA damage and parallels DNA repair. It has also been reported that the reduction of activity of poly (ADP-ribose) polymerase by drugs increases DNA amplification and consequent oncogenesis in cells. Harris, Int. J. Radiat. Biol. 48:675-690 (1985).
In recent years, much work has centered around the exact mechanism by which poly(ADP-ribose)polymerase modulates the DNA replication/repair processes of mammalian cells. Berger et al., pp. 185-195 in Smulson and Sugimura, eds., "Novel ADP-Ribosylations of Regulatory Enzymes and Proteins," Elsevier, N.Y. (1980). It is known that this enzyme is a 113 kDa protein which uses NAD as a substrate in the formation of poly (ADP-ribose) polymerase chains at sites on many nuclear proteins. The enzyme binds tightly to DNA and requires DNA strand breaks for enzymatic activity. Benjamin et al., J. Biol. Chem. 255:10502-10508 (1980). It has been hypothesized that the enzyme system functions in response to transient and localized DNA strand breaks in cells that may arise through a variety of processes including DNA repair, replication, recombination and gene rearrangement. Alkhatib et al., PNAS USA 84:1224-1228 (1987). One reference has taught the measuring of the activity of this enzyme as a method of detecting a predisposition to cancer. Pero, European Patent No. 229,674, published Jul. 22, 1987.
In order to more precisely define the role of poly (ADP-ribose) polymerase, the gene for this enzyme has been sequenced and cloned and localized. Kurosaki et al., J. Biol. Chem. 262:15590 (1987) describes the sequence of cDNA clones representing most of a 4.9 kb mRNA for human poly (ADP-ribose) synthetase from transformed human fibroblasts. The investigators showed the restriction endonuclease map for the cloned cDNAs which reveals two Hind III sites and one Pst I site.
In Cherney et al., Proc. Natl. Acad. Sci. (USA) 84:8370 (December 1987), two of the present inventors and co-authors disclose the first intact cloned cDNA sequence of human poly (ADP-ribose) polymerase. They also disclosed the restriction endonuclease map with two Pst I sites and two Hind III sites. RLFP analysis was done on normal individuals' lymphocytes and fibroblast cell lines to determine chromosome location and two Hind III allele polymorphism were observed on chromosomes in samples from normal patients. It was concluded that the active and expressed gene was on human chromosome one and the processed pseudogene was on chromosome 13.
Thus, the recent cloning of the cDNA for the nuclear enzyme poly (ADP-ribose) polymerase allowed for subsequent derailed sequence analysis of this nuclear protein as well as detailed human chromosomal localization and initial characterizations of the polymorphism of this gene in the normal human population.
A restriction fragment length polymorphism (RFLP) of PADPRP-related sequences was identified and found to correlate with endemic Burkitts lymphoma, B cell follicular lymphoma and lung carcinoma (Bhatia et al., Cancer Res. 50:5406-5413 (1990)). A simple two allele (A/B) polymorphism localized to chromosome 13q33-&gt;qter was noted, in which the frequency of the B allele showed a two to three fold increase when tumor DNA were compared to a non-cancer population (Bhatia et al., Cancer Res. 50:5406-5413 (1990)).
The RFLP was identified by a Hind III 2.7-2.5 kb allelic combination using a human PADPRP cDNA as a probe, and was thought to be either a processed PADPRP pseudogene or a gene with extensive identity to PADPRP. Furthermore, the RFLP did not reflect the gene encoding the authentic PADPRP protein which occurs on chromosome 1 q or the pseudogene on chromosome 14 (Cherney et al., Proc. Natl. Acad. Sci. USA 84:8370-8374 (1987)). A preliminary characterization of this simple two allele (A/B) polymorphism showed that a number of other restriction enzymes including Kpn I, Eco RI, Bgl III, Rsa I and Msp I also identified this polymorphism which always cosegregated together and differed by 200 base pairs (bp) between the respective A and B alleles. Collectively, these RFLP's suggested a deletion or insertion of DNA of at least 200 bp adjacent to, or within PADPRP-like sequences.
In the initial study, analysis of DNA derived from tumor and normal tissue of the same individual revealed that the predominant source of this polymorphism was germline (Bhatia et al., Cancer Res. 50:5406-5413 (1990)). In addition, a tumor derived loss of heterozygosity was noted in 5% of the matched samples, and was not related to a deletion of the retinoblastoma gene (13q14) or other regions of the q arm of chromosome 13. In the noncancer population, a marked difference in the frequency of the B allele was also observed in germline DNA between racial groups (0.14 for whites and 0.35 for blacks). Moreover, an increased frequency of the B allele was still observed in tumor DNA compared to the racially appropriate noncancer germline DNA.
To provide insight into the association between the PADPRP polymorphism on chromosome 13 and a possible predisposition to cancer, we extended the previous survey by studying the B allele frequency from germline derived DNA in a new group of patients with cancers which occur more frequently in the black population (multiple myeloma, prostate and lung cancer). The genomic structure of the polymorphic PADPRP sequences was characterized by cloning and sequencing the Hind III allelic fragment. This report presents data which indicate that the polymorphism reflects a 193 bp duplication of PADPRP processed pseudogene sequences on chromosome 13. In addition, a strategy was developed to analyze the PADPRP genotype of patients using the polymerase chain reaction (PCR), in which the DNA sequences responsible for the A/B polymorphism could be selectively amplified.